Collagenase from smooth weakfish: extraction, partial purification, characterization and collagen specificity test for industrial application / Colagenase de pescada branca: extração, purificação parcial, caracterização e teste de especificidade ao colágeno para aplicação industrial

Fish processing residues are rich sources of biomolecules with industrial potential, such as enzymes with collagenolytic properties applied in the pharmaceutical, textile and leather sectors. Here, collagenolytic serine proteases were partially purified from the waste (viscera) of smooth weakfish Cynoscion leiarchus and characterized for the purpose of obtaining a value-added product from fisheries resources. The higher activity of the enzyme (72.5 U mL-1) was verified in optimal temperature and pH of 55oC and 8.0, respectively. The enzyme was stable in wide ranges of temperature (25-60oC) and pH (6.5 to 11.5). The ions Ca2+ and Mg2+ increased the protease activity, whilst Pb2+, Al3+ and Cu2+ had an inhibitory effect, as observed in the presence of Benzamidine and TLCK (inhibitors of serine proteases). Hydrolysis was detected after 48 hours, when the enzyme and bovine collagen type I were incubated together. Thus, digestive viscera of C. leiarchus is suggested as an alternative source of enzymes capable of cleaving type I collagen, with similar biochemical properties to bacterial collagenases commonly employed in industrial processes, reducing costs, adding value to the fisheries product and minimizing the environmental impact caused by its waste.(AU)

Resíduos do processamento do pescado são fontes ricas em biomoléculas com potencial industrial, como as enzimas com propriedades colagenolíticas empregadas nos segmentos farmacêuticos, têxteis e de couro. No presente trabalho, serino-proteases colagenolíticas dos resíduos (vísceras) do processamento de pescada-branca, Cynoscion leiarchus, foram parcialmente purificadas e caracterizadas, visando à obtenção de um produto de valor agregado, maximizando o aproveitamento de recursos pesqueiros. A atividade da melhor etapa de extração foi 72,5 U mL- 1 , com temperatura e pH ótimos de 55oC e 8.0, respectivamente. A enzima manteve-se estável em faixas amplas de temperatura (25-60oC) e pH (6,5-11,5). Os íons Ca2+ e Mg2+ aumentaram a atividade proteolítica, ao passo que Pb2+, Al3+ e Cu2+ inibiram essa atividade assim como os inibidores de serino-proteases (Benzamidina e TLCK). A hidrólise foi detectada após 48h de incubação com colágeno bovino tipo I. Assim, sugere-se vísceras digestivas de C. leiarchus como fonte alternativa para o fornecimento de enzimas com capacidade de clivar o colágeno do tipo I e com propriedades bioquímicas semelhantes às colagenases bacterianas já empregadas nas etapas de processamento industrial, como forma de redução de custo, agregação de valor ao produto pesqueiro e contribuindo para minimizar o impacto ambiental deste tipo de resíduo.(AU)

Collagenase from smooth weakfish: extraction, partial purification, characterization and collagen specificity test for industrial application / Colagenase de pescada branca: extração, purificação parcial, caracterização e teste de especificidade ao colágeno para aplicação industrial

Fish processing residues are rich sources of biomolecules with industrial potential, such as enzymes with collagenolytic properties applied in the pharmaceutical, textile and leather sectors. Here, collagenolytic serine proteases were partially purified from the waste (viscera) of smooth weakfish Cynoscion leiarchus and characterized for the purpose of obtaining a value-added product from fisheries resources. The higher activity of the enzyme (72.5 U mL-1) was verified in optimal temperature and pH of 55oC and 8.0, respectively. The enzyme was stable in wide ranges of temperature (25-60oC) and pH (6.5 to 11.5). The ions Ca2+ and Mg2+ increased the protease activity, whilst Pb2+, Al3+ and Cu2+ had an inhibitory effect, as observed in the presence of Benzamidine and TLCK (inhibitors of serine proteases). Hydrolysis was detected after 48 hours, when the enzyme and bovine collagen type I were incubated together. Thus, digestive viscera of C. leiarchus is suggested as an alternative source of enzymes capable of cleaving type I collagen, with similar biochemical properties to bacterial collagenases commonly employed in industrial processes, reducing costs, adding value to the fisheries product and minimizing the environmental impact caused by its waste.

Resíduos do processamento do pescado são fontes ricas em biomoléculas com potencial industrial, como as enzimas com propriedades colagenolíticas empregadas nos segmentos farmacêuticos, têxteis e de couro. No presente trabalho, serino-proteases colagenolíticas dos resíduos (vísceras) do processamento de pescada-branca, Cynoscion leiarchus, foram parcialmente purificadas e caracterizadas, visando à obtenção de um produto de valor agregado, maximizando o aproveitamento de recursos pesqueiros. A atividade da melhor etapa de extração foi 72,5 U mL- 1 , com temperatura e pH ótimos de 55oC e 8.0, respectivamente. A enzima manteve-se estável em faixas amplas de temperatura (25-60oC) e pH (6,5-11,5). Os íons Ca2+ e Mg2+ aumentaram a atividade proteolítica, ao passo que Pb2+, Al3+ e Cu2+ inibiram essa atividade assim como os inibidores de serino-proteases (Benzamidina e TLCK). A hidrólise foi detectada após 48h de incubação com colágeno bovino tipo I. Assim, sugere-se vísceras digestivas de C. leiarchus como fonte alternativa para o fornecimento de enzimas com capacidade de clivar o colágeno do tipo I e com propriedades bioquímicas semelhantes às colagenases bacterianas já empregadas nas etapas de processamento industrial, como forma de redução de custo, agregação de valor ao produto pesqueiro e contribuindo para minimizar o impacto ambiental deste tipo de resíduo.

Effect of low-level laser therapy in an experimental model of osteoarthritis in rats evaluated through Raman spectroscopy.

OBJECTIVE: This work aimed to investigate the biochemical changes associated with low-level laser therapy (LLLT) using 660 and 780 nm, on a well-established experimental model of osteoarthritis (OA) in the knees of rats with induced collagenase, using histomorphometry and Raman spectroscopy. MATERIALS AND METHODS: Thirty-six Wistar rats were divided into four groups: control (GCON, n=9), collagenase without treatment (GCOL, n=9), collagenase with LLLT 660 nm treatment (G660, n=8), and collagenase with LLLT 780 nm treatment (G780, n=10). LLLT protocol was: 30 mW power output, 10 sec irradiation time, 0.04 cm(2) spot size, 0.3 J energy, 0.75 W/cm(2) irradiance, and 7.5 J/cm(2) fluence per session per day, during 14 days. Then, knees were withdrawn and submitted to histomorphometry and Raman spectroscopy analysis. Principal components analysis (PCA) and Mahalanobis distance were employed to characterize the spectral findings. RESULTS: Histomorphometry revealed a significant increase in the amount of collagen III for the group irradiated with 660 nm. The Raman bands at 1247, 1273, and 1453 cm(-1) (from principal component score PC2), attributed to collagen type II, and 1460 cm(-1) (from PC3), attributed to collagen type III, suggested that the LLLT causes acceleration in cellular activity, especially on the cells that repair cartilage, accelerating the breakdown of cartilage destroyed by collagenase and stimulating the fibroblast to synthesize repairing collagen III. CONCLUSIONS: LLLT accelerated the initial breakdown of cartilage destroyed by collagenase and stimulated the fibroblast to synthesize the repairing collagen III, suggesting a beneficial effect of LLLT on OA.

Análisis de MMPs en fluidos orales en el diagnóstico complementario de las enfermedades periodontales / Oral-fluid MMP analysis in the complementary diagnosis of periodontal diseases

La periodontitis constituye la infección bacteriana más prevalente a nivel mundial y representa un factor de riesgo para diversas patologías sistémicas. El estado de inflamación y destrucción periodontal se manifiestan a través de la presencia de biomarcadores en el suero y fluidos orales, tales como el fluido gingival crevicular (FGC), saliva y enjuague oral. Enzimas como las metaloproteinasas de matriz (MMP) y mieloperoxidasa, constituyen biomarcadores potenciales para ensayos moleculares complementarios a la clínica de uso en el sillón dental. A continuación se presenta una revisión de la literatura respecto de la aplicación potencial del análisis de metaloproteinasas de matriz extracelular (MMPs) en el diagnóstico complementario de las enfermedades periodontales. Se ha demostrado que los niveles de MMP-9, -13 y particularmente de MMP-8, se asocian con el grado de inflamación periodontal, y pueden diferenciar entre sujetos sanos, con gingivitis, periodontitis y peri-implantitis, mientras que la mejoría de los parámetros clínicos en respuesta al tratamiento periodontal se asocia con la reducción de la activación y niveles de estas enzimas en FGC, como así también en el suero. Se concluye que la determinación, particularmente de MMP-8 en fluidos orales presenta un elevado potencial como complemento de los métodos clínicos tradicionales para identificar a los pacientes con periodontitis o en riesgo de desarrollar la enfermedad, monitorear fases del tratamiento y mejoría de signos periodontales e incluso evaluar el estado de inflamación sistémica.

Periodontal disease is the most common bacterial infection worldwide and it can contribute to enhance the risk for the development of several systemic diseases. The status of periodontal inflammation and destruction can be reflected in biomarker measurement in serum and oral fluids, like gingival crevicular fluid (GCF), saliva and mouth-rinse. Some enzymes, such as matrix metalloproteinases (MMPs) and myeloperoxidase are potential candidates for chair-side point-of-care oral fluid assays. This review is focused on the utility of matrix metalloproteinase (MMP) analysis in oral fluid as a complementary diagnostic method to chronic periodontitis. Levels of MMP-9,-13 and specially of MMP-8, reflect oral inflammatory status and discriminate among healthy, gingivitis, periodontitis and periimplantitis individuals, whereas MMP levels and activation in GCF and serum are in line with the improvement of clinical parameters in response to periodontal treatment. As a conclusion, MMP-8 assessment in GCF could represent a helpful adjunctive method to traditional diagnostics to identify periodontitis or patients at risk to develop the disease, monitor treatment phases, improvement of periodontal signs and even screen the systemic inflammation status.

Capacidad inhibitoria de la actividad proteolítica del Elhibin: su efecto en suero de pacientes quemados / Inhibitory capacity of the proteolytic activity of ELHIBIN: its effects on serum from burned patients

Elhibin es un inhibidor de proteinasas obtenido de semillas de leguminosas. Es capaz de inhibir la elastasa leucocitaria, fibroblástica y también la triptasa. Estas enzimas desempeñan un papel importante en los procesos de envejecimiento e irritación de la piel, por lo tanto, una aplicación específica del Elhibin contribuye a mantener la piel elástica, suave, tersa y húmeda. También contrarresta la inflamación e irritación producto de exposiciones excesivas al sol, agresividad de sustancias químicas y otras influencias ambientales. Teniendo en cuenta estas propiedades se decidió evaluar la capacidad inhibitoria del Elhibin sobre algunas enzimas proteolíticas, utilizando para ello técnicas colorimétricas que emplean la elastina rojo congo y la azocaseína como sustratos naturales específicos, y de manera complementaria la difusión radial. En busca de nuevas aplicaciones para el Elhibin se realizaron ensayos de inhibición de actividad proteolítica en muestras de sueros de pacientes quemados con comportamientos cinéticos o puntuales, de los cuales se conocen que aparecen alteraciones del balance de proteasas y sus inhibidores y el correspondiente descontrol de sus procesos fisiológicos. Los resultados mostraron inhibición de la actividad elastasa y tripsina dependiente de la concentración de Elhibin, no así en el caso de la colagenasa y una aplicación específica para ayudar a contrarrestar desbalances dañinos en las muestras de quemados. Se obtuvo inhibición de la actividad de elastasa y tripsina mayor que 30 porciento en todos los sueros analizados(AU)

H-ras and Nm23-H1 gene expression and proteolytic activity in squamous cell carcinoma of the uterine cervix.

BACKGROUND: The invasive and metastatic potential of malignant cells results from complex interactions of numerous factors not yet fully understood. Genomic alterations such as ras overexpression and nm23-H1 inhibition have been found to be frequently associated with increased invasiveness in various cancers. On the other hand, secretion of different proteinases are necessary for malignant cells to traverse a network of matrix macromolecules, but the relationship between the genomic alterations and the proteolytic phenotype is still unclear. Our aim was to investigate whether the appearance of the proteolytic phenotype had any correlation with the expression of H-ras and nm23-H1 genes in carcinoma of the uterine cervix. METHODS: Twenty-five samples from patients with carcinoma of the uterine cervix at different clinical stages were studied. Cathepsin B1, plasminogen activator, and collagenase activity were assessed in tissue cytosols using specific synthetic oligopeptides as substrates. The expression of H-ras and nm23-H1 was investigated by means of immunohistochemistry and in situ hybridization. RESULTS: Our results showed that cathepsin B1 was the most consistently elevated proteinase, demonstrating a linear correlation with clinical staging. H-ras expression was found elevated in 40% of the cases. Nm23-H1 protein immunoreactivity was positive in 40% of the cases. No correlation was found among H-ras, cathepsin B1 activity, and survival rate. Among cases with high cysteine proteinase activity, a different clinical behavior depending on the expression of Nm23-H1 was observed. The cases with Nm23-H1 protein had a markedly better survival rate than those lacking this protein. In contrast, the absence of Nm23-H1 in association with high cathepsin B1 activity was a clear indicator of a poor prognosis. CONCLUSIONS: These findings suggest a complex interaction between the proteolytic phenotype and the expression of H-ras and nm23-H1 genes in carcinoma of the cervix that influences the clinical behavior of the tumor.

Matrix metalloproteinases 2, 9, and 13, and tissue inhibitors of metalloproteinases 1 and 2 in experimental lung silicosis.

Exposure to silica induces granulomatous lung inflammation evolving to fibrosis through yet unclear pathogenic mechanisms. We examined the expression of extracellular matrix remodeling molecules: collagenase 3, gelatinases A and B, and TIMP-1 and TIMP-2 in experimental lung silicosis. Rats were instilled with 50 mg of silica and sacrificed after 15 and 60 d. At 60 d a significant increase in lung collagen content was found (170.2 +/- 34.4 versus 88.2 +/- 20.8 microgram/mg in controls, p = 0.01). Gelatin zymography of bronchoalveolar lavage fluid (BALF) from 15 and 60 d revealed bands of progelatinase A and progelatinase B, and lung tissue zymograms showed in addition, the active gelatinase A form at 15 d. By in situ hybridization and immunohistochemistry, early silicotic granulomas exhibited intense staining for all matrix metalloproteinases (MMPs) and TIMPs assayed. Labeling was restricted inside granulomas and surrounding areas. Late silicotic granulomas at 60 d showed lower MMP expression than did early lesions, and in highly fibrotic nodules scarce signal was usually found. TIMP-1 and TIMP-2 showed a moderate reduction in 60-d silicotic nodules. These findings suggest that an imbalance in the expression of MMPs and TIMPs may be implicated in extracellular matrix remodeling and basement membrane disruption during experimental lung silicosis.

Expression of metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in schistosomal portal fibrosis.

Focal extracellular matrix degradation morphologically identified in human portal pipestem fibrosis due to Schistosoma mansoni did not express immunohistochemical reactivity for metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (TIMP-1 and TIMP-2). However, when active schistosomal periovular granulomas were present, a strong reactivity for MMP-1, MMP-2, TIMP-1, and TIMP-2 was observed. No reactivity was ever observed for MMP-9. However, the positive pattern of immunohistochemical expression was not seen in old fibrotic periovular granulomas, which were sometimes situated in other areas of the same microscopic section. Positive staining for MMPs and TIMPs was observed at the same time in hepatocytes and within the apical portion of bile duct epithelium. These findings are consistent with the concept that matrix degradation in recent and old fibroses, in addition to differing at the ultrastructural level, also differs in immunohistochemical expression of metalloproteinases and their inhibitors.

Collagenase specificity in chondrosarcoma metastasis.

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection.

Collagenase specificity in chondrosarcoma metastasis

The treatment of some mesenchymal malignancies has made significant gains over the past few decades with the development of effective systemic therapies. In contrast, the treatment of chondrosarcoma has been limited to surgical resection, with the most significant prognostic indicators being surgical margins and histologic grade. We have reported that MMP-1/TIMP-1 gene expression serves to prognosticate for tumor recurrence in this group of patients. This led to the hypothesis that collagenase activity facilitates cell egression from the cartilaginous matrix. In the current study we examine the specificity of collagenase gene expression in archival human chondrosarcoma samples using semi-quantitative PCR. Messenger RNA was affinity extracted and subject to reverse transcription. The subsequent cDNA was amplified using novel primers and quantitated by densitometry. Ratios of gene expression were constructed and compared to disease-free survival. The data demonstrate that the significance of the MMP-1/TIMP-1 ratio as a predictor of recurrence is confirmed with a larger number of patients. Neutrophil collagenase or MMP-8 was observed in only 5 of 29 samples. Collagenase-3 or MMP-13 was observed in all samples but the level did not correlate with disease-free survival. Since the collagenases have similar activity for fibrillar collagens and cleave the peptide in the same location, post-transcriptional regulatory mechanisms may account for the observed specificity. The determination of the MMP-1/TIMP-1 gene expression ratio not only serves to identify those patients at risk for recurrence but may also serve as a novel therapeutic avenue as an adjunct to surgical resection

Expresion de MMP-2 (colagenasa tipo IV) en el carcinoma gastrico / MMP-2 expression (type IV collagenase) in gastric cancer

La MMP-2 (colagenosa tipo IV) es una proteína que pertence a la familia de las metaloproteinas, cuya función está relacionada con la degradación de la matriz extracelular. Su capacidad para degradar el colágeno IV de las membranas basales podría transformala en un agente facilitador de la diseminación neoplásica. Para ver su relación con algunas características clínico-patológicas e inmunohistoquímicas del cáncer gástrico se estudiaron 98 casos de adenocarcinoma de estómago determinado por inmunohistoquímica la presencia de MMP-2 en las células neoplásicas. Los resultados mostraron que había correlación entre la presencia de MMP-2 con el nivel de invasión parietal del tumor (p=0.03) y con la presencia de metástasis en ganglios regionales (p=0.05). En cambio no hubo asociación entre la expresión de MMP-2 con la frequencia por sexo, la localización dentro del estómago, el tamaño tumoral, el tipo histológico, el grado histológico, ni la expresión de las proteínas MIB-1, bcl-2, c-erbB-2 y p53. Tampoco se relacionó con la presencia de recidiva de la enfermedad ni con la sobrevida a los 5 años. (AU)

Expresion de MMP-2 (colagenasa tipo IV) en el carcinoma gastrico / MMP-2 expression (type IV collagenase) in gastric cancer

La MMP-2 (colagenosa tipo IV) es una proteína que pertence a la familia de las metaloproteinas, cuya función está relacionada con la degradación de la matriz extracelular. Su capacidad para degradar el colágeno IV de las membranas basales podría transformala en un agente facilitador de la diseminación neoplásica. Para ver su relación con algunas características clínico-patológicas e inmunohistoquímicas del cáncer gástrico se estudiaron 98 casos de adenocarcinoma de estómago determinado por inmunohistoquímica la presencia de MMP-2 en las células neoplásicas. Los resultados mostraron que había correlación entre la presencia de MMP-2 con el nivel de invasión parietal del tumor (p=0.03) y con la presencia de metástasis en ganglios regionales (p=0.05). En cambio no hubo asociación entre la expresión de MMP-2 con la frequencia por sexo, la localización dentro del estómago, el tamaño tumoral, el tipo histológico, el grado histológico, ni la expresión de las proteínas MIB-1, bcl-2, c-erbB-2 y p53. Tampoco se relacionó con la presencia de recidiva de la enfermedad ni con la sobrevida a los 5 años.

Metalloproteinase activity during growth, maturation and atresia in the ovarian follicles of the goat.

Metalloproteinases are an important group of hydrolytic enzymes which participate in interstitial matrix degradation during tissue remodelling processes and therefore may be required during follicular growth and maturation. The activity of metalloproteinases (collagenases, gelatinase, and Pz-peptidase), was measured during growth, maturation and atresia of goat antral follicles. These follicles (n = 67) were separated by size and also classified into four groups: non-atretic (Group I); early atretic (Stage I) (Group II); moderately atretic (Stage II) (Group IIIa); and, late atretic (Stage III) (Group IIIb). Pz-peptidase was greater in granulosa than in thecal cells, and almost absent in follicular fluid. In non-atretic follicles, activity in granulosa cells increased with increasing follicle size, whereas activity peaked in 3-6 mm follicles in thecal cells. Atresia was associated with declining activity in thecal cells from follicles in the 3-6 mm range and in granulosa cells from the > 6 mm range. Interstitial collagenase activity was significant and similar in granulosa and thecal cell extracts and low in follicular fluid from non-atretic follicles. Activity increased significantly in thecal cells, but decreased significantly in granulosa cells from large (> 6 mm) non-atretic follicles. Atresia was associated with declining activity in both types cells and increasing activity in follicular fluid. Gelatinase activity was some times associated with five regions corresponding to molecular weights of 22.1, 30.7, 39.6, 63.8 and 71.4 kDa, and rarely at 91.3 and 81.2 kDa. Overall activity declined with atresia in thecal cells from follicles in the 3-6 mm range, but not in those > 6 mm. In granulosa cells from follicles 3-6 mm, activity varied widely with stage of atresia, while in cells from follicles > 6 mm, activity was greatly increased in atretic follicles.

[Dietetic factors and premature rupture of fetal membranes. Effect of vitamin C on collagen degradation in the chorioamnion]. / Factores dietéticos y ruptura prematura de membranas. Efecto de la vitamina C en la degradación de colágena en el corioamnios.

Some studies have shown an association between vitamin C disposability (Vit C), and the development of premature rupture of membranes (RPM). However, vitamin C role in the metabolism of collagen upon chorioamnion tissue, has not been analyzed. In this study the effect of modulation with different vit C concentrations in culture cells derived from human amnion, was analyzed. Vit C concentrations were used in order to cover physiological range (29.0 micrograms/ml). After stimulation the cells media were analyzed for enzymatic activity of metalloproteinases with extracellular matrix (MMP), and relative quantity of MMP-1, MMP-2 and MMP-9, was quantified, by immune transference, using monospecific polyclonal antibodies. The activity, as well as protein decreased in amniotic cells media, in a direct way as to vit C concentration, so, at the highest used concentrations (100 micrograms/ml), the least MMP activity/quantity, was obtained. These results show a finding not described until now, which permits to establish a direct connection between vit C availability and increase in collagen degradation. According to results, the less availability of vit C, the greater degradation of collagen, which should lead to a mechanical support loss and eventual fetal membranes rupture.

[Transition from the latent to the active enzymatic form as a model of regulation of extracellular matrix degradation in the chorioamnion during human labor]. / Transición de la forma enzimática latente a activa como un modelo de regulación de la degradación de matriz extracelular en el corioamnios durante el trabajo de parto humano.

Matrix metallo proteinases (MMP) are the physiological mediators of collagen degradation and its participation in physiopathogenesis of premature rupture of membranes has been suggested by our group. With the idea of defining if some MMP become active active in a coordinated way with labor in fetal membranes, we analyzed enzymatic activity and immunoreactive protein present in extracts of amnion and chorion. It was possible to identify the presence of MMP-9 in extracts of membranes obtained during cesarean sections, without labor, although its activity/quantity was faintly detectable. Instead, extracts of fetal membranes obtained during active labor showed large activity/quantity of this MMP. With a monoclonal antibody, it was possible to show that the active form of MMP-9 could only be found in samples with labor. MMP-9 and its messenger RNA, were localized by immunohistochemistry and in situ hybridization in amniotic epithelium, in some fibroblasts of the compact layer and in trophoblast-like cells in chorion. It is concluded that: 1. Activity and quantity of MMP-9 increase selectively associated to labor; and 2. That this enzyme is expressed by different cellular populations of fetal membranes.